Abstract
The actin cytoskeleton of trabecular meshwork (TM) cells is a therapeutic target for lowering intraocular pressure (IOP) in glaucoma patients. Netarsudil (the active ingredient in RhopressaTM) is a Rho-associated protein kinase inhibitor that induces disassembly of actin stress fibers. Here, we used live cell imaging of SiR-actin-labeled normal (NTM) and glaucomatous TM (GTM) cells to investigate actin dynamics during actin-driven biological processes with and without netarsudil treatment. Actin stress fibers were thicker in GTM than NTM cells and took longer (>120 min) to disassemble following addition of 1 µM netarsudil. Actin-rich extracellular vesicles (EVs) were derived by two mechanisms: exocytosis of intracellular-derived vesicles, and cleavage of filopodial tips, which detached the filopodia from the substratum, allowing them to retract to the cell body. While some phagocytosis was noted in untreated TM cells, netarsudil potently stimulated phagocytic uptake of EVs. Netarsudil treatment induced lateral fusion of tunneling nanotubes (TNTs) that connected adjacent TM cells; TNTs are important for TM cellular communication. Together, our results suggest that netarsudil may clear outflow channels in TM tissue by inducing phagocytosis and/or by modulating TM communication via EVs and TNTs. These cellular functions likely work together to regulate IOP in normal and glaucomatous TM.
Highlights
Trabecular meshwork (TM) cells in the anterior of the eye play a key role in the regulation of intraocular pressure (IOP) [1,2]
This time interval was chosen in pilot studies because 24 fields could be imaged at a time and there was minimal loss of specific SiR-actin labeling due to bleaching, After 60–90 min of no treatment, 1 μM netarsudil or 1 μM Y27632 were added to an injection port and imaging was resumed
In this study, we investigated the effects of the Rho kinase inhibitor, netarsudil, on actin-driven biological processes in live normal and glaucomatous TM cells
Summary
Trabecular meshwork (TM) cells in the anterior of the eye play a key role in the regulation of intraocular pressure (IOP) [1,2]. The actin cytoskeleton provides mechanical support to cells and regulates cellular protrusions, adhesions, and contraction [3] It is highly dynamic, with the constant addition and removal of actin subunits to filamentous actin (F-actin), which is regulated by a myriad of actin-binding proteins [4]. Phosphorylation of myosin light-chain phosphatase by Rho-associated kinase (ROCK) mediates actin stress fiber assembly and cellular contractility [5]. Inhibition of this Rho-ROCK signaling pathway is an effective method to increase aqueous humor outflow and lower IOP [6,7,8,9,10,11]. We observed that parallel TNTs, drawn out when cells retracted from one another, can fuse laterally with netarsudil treatment
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